Methods for the immobilization of biologically active materials, especially enzymes, have undergone such rapid development in recent years that it is fair to say that support matrices and their preparation are rather mature fields of knowledge in, for example, enzyme catalyzed reactions of commercial importance. The impetus for their development initially was the conservation of enzymes; the use of enzymes in homogeneous reactions generally mandated the single use of enzymes. Because enzymes often are an expensive, and perhaps the most expensive, component of reactions there arose the need to develop methods allowing multiple use of enzymes. Immobilization of enzymes on solid supports led to heterogeneous enzyme-catalyzed reactions where the immobilized enzymes could be readily removed, as in a stirred batch reactor, or could be employed in a continuous process, as in a fixed bed, but in either case permitted enzyme-catalyzed processes where the enzyme could be reused until its decreased activity made further use economically unfeasible.
Presently there are a variety of support matrices from which immobilized enzymes specifically, and immobilized biologically active materials generally, can be prepared. Some bind the enzyme, as exemplary of a biologically active material, via ionic interaction, others bind the enzyme via entrapment. In still others the biologically active material is immobilized by covalent bonding to the support or some intermediary linked to the support. Thus, the skilled worker has some realistic alternatives in his technological closet when seeking a support matrix with which to immobilize a biologically active substance.
Nonetheless, there remains some technologically significant gaps in the field of support matrices. One highly desirable goal is the preparation of a support matrix which is conveniently and cheaply regenerable when the activity of its immobilized biologically active material strips it of economic benefit. It is even more preferable that the support matrix could be regenerated multiple times, with its subsequent activity in immobilizing biological material undiminished. Some limited success has been achieved, as e.g., the methods taught in U.S. Pat. Nos. 4,248,969 and 4,250,260, but even more economical systems are greatly desired.
Among the supports that have been used to immobilize enzymes is included diethylaminoethyl cellulose (DEAE-cellulose), a support material chosen because of its relatively low cost but whose use is largely limited to a stirred tank reactor. Kirk-Othmer, "Encyclopedia of Chemical Technology" Third Edition, V. 9, p. 155(J. Wiley & Sons, 1980). Such a support material is unsuitable for use in a packed bed reactor because of its poor flow characteristics, one of the required characteristics necessary for use in a packed bed. In fact, DEAE-cellulose appears to have been used in the first commercial process in the United States using immobilized glucose isomerase (op. cit., p. 157), although it was soon succeeded by other immobilized glucose isomerase systems which could be used as a packed bed.
To attain the good flow necessary for a continuous process in a packed bed reactor it is desirable for the particles to be incompressible, hard, unreactive materials, as are the refractory inorganic oxides such as alumina, silica, glass, and so forth, and ceramics. In fact, the latter materials are core materials in supports where they are coated with an organic resin which binds to the biologically active materials, as exemplified by U.S. Pat. No. 4,141,857.
This application describes a support matrix having all the characteristics necessary for its successful use in a packed bed, but with the additional characteristic of being readily regenerable from an exhausted or deactivated enzyme immobilized thereon. The support matrix of this invention is easy to make, economical, immobilizes a broad variety of biologically active materials, and is regenerable using extraordinarily simple and rapid means.